Identification And Denitrification Properties Of An Aerobic Denitrifying Bacterial Strain Isolated From Aquaculture Pond

In recent years,aquaculture industry in China has developed rapidly.The pollution of aquaculture water has gradually increased with the expansion of high-density aquaculture.Ammonia,nitrate,nitrite,and sulfide are the main pollutants that cause the deterioration of water,which will not only cause toxic effects to aquatic animals,but also result in serious environmental problems,such as eutrophication.At present,one of the most efficient methods to remove nitrogen from water is biological treatment,and denitrifying bacteria play an important role in the biological process.In this study,a safe and indigenous aerobic denitrifying bacterium with high efficiency of nitrogen and sulfur removal was screened from the soil samples of intensive aquaculture ponds.It was identified by 16S r DNA sequence,and its denitrification properties and application in simulative fishery water were also studied.The results and conclusion are as follows.In the research,two aerobic denitrifying strains,named as strains SC16 and SC17,were obtained by enrichment and bromothymol blue plate method from sediments sampled from aquaculture ponds.Based on the results of morphological observation and 16S r DNA gene sequence analysis,these two bacteria were all identified as Bacillus sp..The two strains obtained were both Gram-positive bacteria.Both strains possess ability to produce gas by nitrate reduction and can be used as the alternative strains to eliminate pollutant nitrogen in water.The hemolytic and virulence test indicated that strain SC16 is a non-pathogenic microorganism and non-toxic.The results of removing nitrogen and desulfurization showed that strain SC16 could remove nitrate at 97%,ammonia at 36.6%,nitrite at 99.99%and sulfide at 99.86%when the strain was incubated with nitrate at an initial concentration of 306.9 mg/L for 72 h,ammonia at 165.49 mg/L for 48 h,nitrite at 200 mg/L for 48 h,and sulfide at 70.9 mg/L under aerobic condition.Moreover,the strain strongly tolerates the nitrite at the high concentration of250 mg L^-1.When strain SC16 were added into aquaculture wastewater with the inoculation of 1%（v/v）,the ammonia nitrogen at an initial concentration of 8.35 mg/L,the nitrite at an concentration of 0.45 mg/L and the nitrate at an initial concentration of 0.46 mg/L in the aquaculture wastewater were removed at the cumulative removal rate of 47.6%for ammonia nitrogen,23.9%for nitrate for 11 days,and the concentration of nitrite nitrogen fell below the secure concentration（0.1 mg/L）.The results of denitrification experiments showed that environmental factors such as carbon source,C/N,temperature and pH could affect the rate of removing nitrogen of strain SC16.The maximum bacterial optical desity at 600 nm(OD₆₀₀)could reach 0.33 when citric acid served as carbon source,carbon-nitrogen ratio was 8:1,pH was 7 and the temperature was 28°C,the highest removal rate of nitrate nitrogen of 99.9%was achieved for 24 hours.The sequence of encoding region of the nitrite reductase（nirK）of strain SC16 was obtained by Genome Walking method.The encoding region was 876 bp in length encoding 291 amino acids.During the removal of nitrate,ammonia,and nitrite by strain SC16,the m RNA expression level of nirK was up-regulated,and the transcriptional expression pattern of the nirK was identical with the elimination pattern of nitrate,ammonium,and nitrite in the process of denitrification.It is indicated that the nirK is closely related to degradation of ammonia,nitrate and nitrite.